In this application, the following abbreviations will be used:
NR, non-repetitive; Apr, ampicillin resistance gene; IPTG, Isopropyl-β-D-thiogalactosid; GdmCI, guanidinium chloride; GdmSCN, guanidinium thiocyanate; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; Tris, Tris(hydroxymethyl)aminomethane; CD, circular dichroism; rep-proteins, repetitive proteins; Da, Dalton; cps, counts per second; MRW, mean residue weight; n.d., not determined.
Spider silks are protein polymers that display extraordinary physical properties (1). Among the different types of spider silks, draglines are most intensely studied. Dragline silks are utilized by orb weaving spiders to build frame and radii of their nets and as lifelines that are permanently dragged behind. For these purposes high tensile strength and elasticity are required. The combination of such properties results in a toughness that is higher than that of most other known materials (1;2). Dragline silks are generally composed of two major proteins whose primary structures share a common repetitive architecture (3;4).
Variations of a single repeat unit, which can comprise up to 60 amino acids, are iterated several times to represent the largest part of a dragline silk sequence. These repeat units comprehend a limited set of distinct amino acid motifs. One motif found in all dragline silk repeat units is a block of typically 6-9 alanine residues. In silk threads several poly-alanine motifs form crystalline β-sheet stacks leading to tensile strength (5;6).
Glycine rich motifs such as GGX or GPGXX adopt flexible helical structures that connect crystalline regions and provide elasticity to the thread (7).
Additionally, all investigated dragline silk proteins comprise regions at their carboxyl termini that display no obvious repetition pattern (non-repetitive- or NR-regions). So far no function could be assigned to these regions in the final thread.
Silk assembly in vivo is a remarkable process. Spider dragline silk proteins are stored at concentrations up to 50% (w/v) (8) in the so-called major ampullate gland. Although a “dynamic loose helical structure” has been proposed for the proteins within the major ampullate gland (8) more recent data suggests a random coil conformation for the proteins of the so called A-Zone, which represents the largest part of the gland (9;10). The highly concentrated protein solution forms the silk dope (spinning solution), which displays properties of a liquid crystal (11-13).
Thread assembly is initiated during a passage of the dope through the spinning duct accompanied by extraction of water, sodium and chloride (14;15). At the same time the concentrations of the more lyotropic ions potassium and phosphate are increased and the pH drops from 6.9 to 6.3 (14-16). Assembly is finally triggered by mechanical stress, which is caused by pulling the thread out of the spider's abdomen (17).
For several purposes natural silk threads can not be used directly, but have to be dissolved and reassembled into other morphologies such as films, foams, spheres, nanofibrils, hydrogels and the like.
Most investigations concerning films made from silk proteins have been performed with silk fibroin, the main protein component of the silk from the silkworm Bombyx mori. Silk fibroin films can be cast from aqueous solutions or from solutions containing hexafluoroisopropanol (HFIP), formic acid, and trifluoro acetic acid. In solution silk fibroins tend to adopt helical or random coil conformations, depending on the solvent used. When cast into films, proteins either maintain the conformation of the soluble state or adopt a more β-sheet rich conformation. In most cases processing of the films with methanol leads to a further increase of β-sheet content and crystallinity. Besides silk fibroin, other silk proteins have also been employed to cast films. Vollrath and co-workers investigated films made of proteins extracted from major ampullate gland of the spider Nephila senegalensis. As-cast films mainly contained proteins in a random coil conformation when prepared from aqueous solution. Their structure changed into β-sheet upon addition of potassium chloride. Further, films have been made from a synthetic silk protein derived from the dragline silk protein MaSp1 of the spider Nephila clavipes using HFIP as solvent. In solution the protein adopted an α-helical structure changing to a more β-sheet rich conformation when cast into a film.
Unfortunately, the generation of functional film materials from natural silk fibroin is restrained by its amino acid sequence. Selective chemical modification of silk fibroin is only possible to a very limited extend due to the low abundance (<1.5%) of chemically reactive amino acid side chains that contain thiol, amino or carboxyl groups. Further, genetic modification within the natural host to alter the silk protein's and thus the film's properties is tedious.
While some structural aspects of spider silk proteins have been unravelled, still little is known about the contribution of individual silk proteins and their primary structure elements to the assembly process. Comparative studies of the two major dragline silk proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, revealed that, although their amino acid sequences are rather similar (4), they display remarkably different solubility and assembly characteristics: While ADF-3 is soluble even at high concentrations (18), ADF-4 is virtually insoluble and self-assembles into filamentous structures under specific conditions (unpublished results).
Scientific and commercial interest initiated the investigation of industrial scale manufacturing of spider silk. Native spider silk production is impractical due to the cannibalism of spiders, and artificial production has encountered problems in achieving both sufficient protein yield and quality thread-assembly. Bacterial expression yielded low protein levels, likely caused by a different codon usage in bacteria and in spiders. Synthetic genes with a codon usage adapted to the expression host led to higher yields, but the proteins synthesized thereof showed different characteristics in comparison to native spider silks. Expression of partial dragline silk cDNAs in mammalian cell lines did yield silk proteins (e.g. ADF-3) that could be artificially spun into ‘silken’ threads, albeit as yet of inferior quality.
WO03060099 relates to methods and devices for spinning biofilament proteins into fibers. This invention is particularly useful for spinning recombinant silk proteins from aqueous solutions and enhancing the strength of the fibers and practicality of manufacture such as to render commercial production and use of such fibers practicable. Therein, it is disclosed to express spider silk proteins in mammalian cells, e.g. transgenic goat mammary gland cells.
Expression of authentic spider silk genes in bacterial hosts is—as mentioned above—inefficient (24) since some gene sections contain codons not efficiently translated in bacteria. In addition, gene manipulation and amplification by PCR is difficult due to the repetitive nature of silks. In order to investigate properties of spider silk proteins, cloning strategies have been employed using synthetic DNA modules with a codon usage adapted to the corresponding expression host. Synthetic genes were obtained which coded for proteins resembling the repetitive regions of spider silks (25-28). However, none of these protein designs included the carboxyl terminal NR-regions that are found in all dragline silks.